RNAi therapeutics are emerging new ways to combat human diseases through silencing of undesired gene expressions. The discovery of long double-stranded RNA mediated RNAi in the worm (Fire, A. et al. Nature 1998; 391:806-811) followed by demonstration of RNAi mediated by small interfering RNA (siRNA) in mammalian cells (Elbashir, S. M. et al. Nature 2001; 411:494-498) have generated an unprecedented global interest in RNAi therapeutics. The small RNA molecules involved in RNAi pathways include small interfering RNAs (siRNAs) and microRNAs (miRNAs) with the latter deriving from imperfectly paired non-coding hairpin RNA structures those are naturally transcribed by the genome (Meister, G. and Tuschi, T. Nature 2004; 431:343-349; Kim, D. H. and Rossi, J. J. Nature Rev Genet 2007; 8:175-184). siRNA mediates gene silencing through sequence specific cleavage of perfectly complementary messenger RNA (mRNA) whereas gene silencing by miRNAs are mediated through translational repression and transcript degradation for imperfectly complementary target messenger RNAs. The steps involved in the endogenous production of microRNAs include: (a) processing of RNAs with stems or short-hairpin structures (encoded in the intragenic regions or within the introns) in the nucleus to form precursor RNA molecules called pre-microRNAs; (b) export of the pre-microRNAs from the nucleus into the cell cytoplasm; (C) further shortening and processing of the pre-miRNAs by an RNase III enzyme called Dicer to produce an imperfectly matched, double-stranded miRNA (Kim, D. H. and Rossi, J. J. Nature Rev Genet 2007; 8:175-184; He, L. and Hannon, G. J. Nature Rev Genet 2004; 5:522-531). Dicer similarly processes long, perfectly matched dsRNA into siRNAs. A multi-enzyme complex including the Argonoute 2 (AGO2) and the RNA-induced silencing complex (RISC) binds to either the microRNA duplex or the siRNA duplex and discards one strand forming an activated complex containing the guide or antisense strand (Mantranga, C. et al. Cell 2005; 123:607-620). The activated AGO2-RISC complex then induces silencing of gene expression by binding with the mRNA strand of complementary sequence followed by its subsequent cleavage. Gene silencing through mRNA cleavage owes its potency to the rapid nucleolytic degradation of the mRNA fragments. Once the mRNA is degraded, the activated RISC complex becomes free to bind and cleave another target mRNA in a catalytic fashion (Hutvagner, C and Zamore, P. D. Science 2002; 297:2056-2060). The first in vivo study on RNAi-based therapeutics was disclosed in an animal disease model in 2003 (Song, E. et al. Nat. Med. 2003; 9:347-351). Ever since then, a plethora of in vivo studies on RNAi therapeutics have been reported. siRNA mediated inhibitions of vascular endothelial growth factor have been demonstrated to be capable of suppressing tumor vascularization and growth in mice (Filleur, S. et al. Cancer Res. 2003; 63:3919-3922, Takei, Y. et al. Cancer Res. 2004; 64:3365-3370) as well as in inhibiting ocular neovascularization in a mouse model (Reich, S J et al. Mol. Vis. 2003; 9:210-216). Galun, E. demonstrated that replication of hepatitis B virus in mice can be inhibited by siRNA (Mol. Ther. 2003; 8:769-776). Small interfering RNA directed against beta-catenin has been shown to inhibit the in vitro and in vivo growth of colon cancer cells (Verma, U N et al. Clin. Cancer Res. 2003; 9:1291-1300). Caspase 8, small interfering RNA has been shown to be capable of preventing acute liver failure in mice (Zender, L. et al. Proc. Natl. Acad. Sci. USA. 2003; 100:7797-7802) Inhibition of influenza virus production in virus-infected mice has been achieved through RNA interference (Ge, Q. et al. Proc. Natl. Acad. Sci. USA. 2004; 101:8676-8681, Tompkins, S M et al. Proc. Natl. Acad. Sci. USA. 2004; 101:8682-8686). Use of siRNA targeting Fas has been used to protect mice against renal ischemia-reperfusion injury (Hamar, P. et al. Proc. Natl. Acad. Sci. USA. 2004; 101:14883-14888). Small interfering RNA, upon nasal administration, has been shown to inhibit respiratory viruses (Bitko, V. et al. Nat. Med. 2005; 11:50-55). siRNA targeting Raf-1 can inhibit tumor growth both in vitro and in vivo (Leng, Q. and Mixson, A J. Cancer Gen. Ther. 2005; 12:682-690). Small interfering RNA against CXCR-4 blocks breast cancer metastasis (Liang Z. et al. Cancer Res. 2005; 65:967-971). Intravesical administration of siRNA targeting PLK-1 successfully prevented the growth of bladder cancer (Nogawa, M. et al. J. Clin. Invest. 2005; 115:978-985). Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1 has been achieved (Shen, J. et al. Gene Ther. 2006; 13:225-234). Selective gene silencing in activated leukocytes has been demonstrated by targeting siRNA to the integrin lymphocyte function-associated antigen (Peer, D. et al. Proc. Natl. Acad. Sci. USA. 2007; 104:4095-4100). Recently, RNAi in human has been demonstrated (Davis, M. E. et al. Nature 2010; 464:1067-70).
Cell division cycle 20 (Cdc20) is an essential cell cycle regulator required for the completion of mitosis in organisms from yeast to human. In the cell cycle, activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis. Cdc20 is one of the key regulators for APC; it binds to APC and activates its cyclin ubiquitination activity (Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Nature, 349, 132-138). This complex recognizes a 9 amino acid sequence (destruction box [D-box]) in the N terminus of cyclin and a few other proteins, catalyzing the transfer of ubiquitin from the thioester of a UBC-ubiquitin complex to a free amino group on the protein and then linking further ubiquitins to the ubiquitinated substrate to generate a large chain of ubiquitins; the tagged protein is then recognized by the proteasome and degraded (King, R W. et al. (1995) Cell, 81, 279-288. Aristarchus, A. et al. (1996) Proc. Natl. Acad. Sci. USA, 93, 4294-9). In metaphase, sister chromatids cannot separate as they are held together at their centromeres and along the chromosome arms by multiprotein complex of SMC family, named cohesins. Again, the SMC cohesion proteins are associated by Sccl protein. Before anaphase, securin binds to and inhibits separase, a ubiquitous protease. At the onset of anaphase, when all the chromosome kinetochores have arranged in the spindle microtubules, APC/Cdc20 complexes polyubiquitinates securin. Thus, securin is degraded by proteases and the free separase now cleaves Sccl making the sister chromatid free. The pole ward force exerted on kinetochores pulls the sister chromatids towards the opposite spindle poles. Since Cdc20 is highly expressed in several carcinomas [Hamada, K. et al. (2004) Cancer Gen Prot 1: 231-240, Kim, J M. et al. (2005). Clin Cancer Res 11: 473-482.], knockdown of the expression of Cdc20 through RNA interference holds therapeutic promise in combating cancer. Recently, at the cellular level, it has been reported that Cdc20 siRNA can inhibit more than 90% Cdc20 expression at both the transcriptional and translational levels and the specific knockdown of Cdc20 expression inhibited the cell growth of human pancreatic carcinoma cells in vitro [Taniguchi, K. et al. (2008). Anticancer Res 28: 1559-1563].
Guanidinylated cationic amphiphiles are, in general, efficient in delivering genetic materials into cells. Many distinguishing factors contribute to the high gene transfer efficiencies of guanidinylated cationic amphiphiles. The guanidinium head-groups remain protonated over a much wider range than other basic groups due to its remarkably high pka values (13.5) and therefore they strongly bind with polyanionic macromolecular DNA molecules under the physiological pH; in addition to forming electrostatic complexes with genetic materials, they form characteristic parallel zwitterionic N—H+ . . . O—hydrogen bonds with the phosphate ions of the nucleotides and they are capable of forming hydrogen bonds with nucleic acid bases [Vigneron, J P. et al. (1996). Proc. Natl. Acad. Sci., USA 93: 9682-9686.]. Previously we have demonstrated that the guanidinylated cationic amphiphile with myristyl (n-C14H29) tail (lipid 1, FIG. 1a) is most efficient in delivering reporter gene into cultured animal cells at lipid:DNA charge ratio of 3:1 and 1:1 [Sen, J, Chaudhuri, A. (2005). J Med Chem 48: 812-820.].
Beyond identifying an active target sequence, a key challenge in the field of RNAi therapeutics is ensuring efficient delivery of small interfering RNAs inside the cell cytoplasm. Efficient intracellular delivery of biologically active compounds have previously been accomplished using liposomes, microscopic fatty bubbles of amphiphilic molecules which contain both hydrophobic (water hating) and hydrophilic (water loving) regions in their molecular architectures. Several methods for complexing biologically active compounds with liposomes have been developed. For instance, DOTMA (N-1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was the first cationic amphiphile used to deliver biologically active polynucleotides (Felgner et al. Proc. Natl. Acad. Sci. USA. 1987; 84:7413-7417). Ever since then, a plethora of cationic amphiphiles have been used in delivering polynucleotides into the cell cytoplasm (Karmali, P. P. and Chaudhuri, A. Med. Res. Rev. 2007; 27:696-722 and the references cited therein). Cationic liposomes in particular, are least immunogenic. Manufacturing a greater degree of control can be exercised over the lipid's structure on a molecular level and the products can be highly purified. Use of cationic liposomes does not require any special expertise in handling and preparation techniques. Cationic liposomes can be covalently grafted with receptor specific ligands for accomplishing targeted gene delivery. Such multitude of distinguished favorable clinical features are increasingly making cationic liposomes as the non-viral transfection vectors of choice for delivering polynucleotide into body cells.